The past decades have witnessed a dramatic increase in the prevalence of obesity and type-2-diabetes (T2D) primarily in US but also progressing in Europe and in developing countries. All over the world, T2D represents 85-90% of all the cases of diabetes (inherited insulin-dependent diabetes or T1D and non-insulin-dependent-diabetes Mellitus or T2D) (King et al., 1998). Although several mutations affecting leptin pathway, insulin secretion or its receptor, or GLUT4 (glucose transport sensitive to insulin) have been identified, most of the cases of obesity and T2D are from non-genetic origin and triggered by life style (reduction of physical activity, highly calorie diets, etc). T2D and obesity are both associated with high morbidity, mortality (Stiegler et al., 1992), and health care costs (Rubin et al., 1994).
The primary feature of T2D is insulin resistance and defect in insulin secretion. Insulin is a key hormone synthesized and secreted by pancreatic beta-cells, that stimulates glucose uptake in various organs (particularly muscle, liver, and adipose tissue). Insulin also regulates Hepatic Glucose Production (HGP) via controlling the expression of the gene encoding glucose-6-phosphatase and inhibits lipolysis in adipose tissue (Murray et al., 2000). Impaired insulin action (i.e. insulin resistance) occurs when target tissues are unable to respond to normal concentrations of insulin.
Once this deregulation starts and in absence of treatment, beta-cells secrete increased amount of insulin (=hyperinsulinemia) to maintain euglycemia (normal circulating glucose levels). However, in the absence of treatment, beta-cells fail producing enough insulin, leading to increase in circulating glucose (hyperglycaemia) (FIG. 1). As long as enough beta-cells will be viable and will be secreting the appropriate rate of insulin to maintain euglycemia, T2D does not arise (Kahn, 1998). The mechanisms leading to insulin resistance have been extensively explored during the past years by many groups. It is widely accepted that the accumulation of free fatty acids (FFA) in insulin-sensitive non adipose tissues (liver & muscles), can impair insulin-mediated-glucose uptake in these tissues. (Randle et al., 1963). Moreover, increased lipid production by the liver enhances fatty acid oxidation, decreases insulin-dependent inhibition of hepatic glucose production and, therefore, increases gluconeogenesis (GNG), further worsening the hyperglycaemia (Boden, 2003).
Development of T2D is associated with other metabolic disturbances. The cluster of insulin resistance, impaired glucose tolerance, arterial hypertension, abdominal obesity and dyslipidemia, called metabolic syndrome (Syndrome X) (Reaven, 1988), has been defined by the Adult Treatment Panel III (ATPIII) as a grouping of factors that underlie major cardiovascular risk (Grundy et al., 2004). Many preclinical studies suggested a predominant role of insulin in the development of hypertension in T2D patients (Scherrer et al., 1997). However, the primary focus of clinical care is to diagnose and treat the abnormalities in glucose metabolism. Indeed high blood glucose is a major risk factor for microvascular complications as reported by the UK Prospective Diabetes Study (UKPDS, 1998). It was shown that maintenance of glucose levels near to normal in T2D patients prevents the onset of defects such as neuropathy (occurring in 50% to 60% of T2D patients), retinopathy and nephropathy (diabetes are the leading causes of blindness and end-stage renal failure in the U.S) (Klein, 1995; Teutsch et al., 1989).
Some years ago, non-pharmacological therapy took place in the US, based on diet, exercise and weight loss (Tinker et al., 1994). These major lifestyle modifications did not only lower blood glucose concentration, but also reduced or delayed occurrence of risk factors for cardiovascular diseases (CVD) in overweight diabetic patients (Schneider et al., 1995). However, diabetic patients with advanced disease require specific medication to control their glycaemia and to substantially prevent or reduce appearance of complications. Current anti-diabetic therapy is based on a tight control of circulating glucose by either (1) improving insulin production by the use of agents commonly known as insulin secretagogues or (2) improving whole body insulin action with or without inhibition of hepatic glucose production by agents known as insulin sensitizers (FIG. 1).
The anti-diabetic drugs from the thiazolidinediones (TZDs) class (available in the US since 1997), currently represented by Rosiglitazone (Avandia®), Pioglitazone (Actos®), and Troglitazone (Rezulin®), are efficacious drugs in increasing peripheral insulin-mediated-glucose-uptake. TZDs are pharmacological agonists of peroxisome-proliferator-activated receptor (PPAR gamma), a transcription factor of the nuclear hormone receptor family that controls the expression of genes in glucose and lipid metabolism. PPAR gamma drugs reduced hyperglycaemia, hyperlipidemia and hyperinsulinemia and improved insulin sensitivity by increasing differentiation and proliferation of pre-adipocytes into mature fat cells, particularly in peripheral fat depots (Gurnell et al., 2003). Thus PPAR gamma activation increases fatty acids storage in peripheral adipocytes, lowers circulating fatty acids and reduces triglycerides levels in muscle and liver. PPAR gamma drugs alter the expression of several circulating factors such as adiponectin, TNF alpha and resistin, the levels of which are highly correlated to insulin resistance and the response to therapy (Greenfield et al., 2004).
Appropriate animal models of T2D and insulin resistance are essential preclinical tools for characterizing in vivo efficacy of therapeutic agents. Most of the animal models of T2D that have been developed in the past 20 years are genetic based. Spontaneously diabetic (or insulin resistant and obese) rodent models such as, db/db and ob/ob mice, GK, ZDF and fa/fa rats are most commonly used worldwide in drug discovery (Chen et al., 2004) (Table 1). Among these animal models, Zucker diabetic fatty (ZDF) rats when fed with diabetogenic diet (Purina 5008 or KLIBA 2437) represent the most attractive model since metabolic disorders (glucose intolerance, hyperglycaemia, insulin resistance and hypertriglyceridemia), beta-cell failure, obesity and mild hypertension develop similarly to humans, although in a more rapid progression. Early changes in main plasma parameters start at 7-8 weeks of age leading to overt diabetes (beta-cell and renal failure) at >=12 weeks of age (FIG. 2).
Due to this rapid metabolic deterioration, reversion of main feature of T2D in ZDF rats is not achieved by marketed anti-diabetics (Rosiglitazone (PPAR gamma) and Raziglitazar (PPAR alpha gamma)). Prevention of T2D was shown to be achieved, however, in ZDF rats under chronic treatment (13 weeks) (Shibata et al., 2000) or in very young and only moderately diabetic animals (Brand et al., 2003; Pickavance et al., 2005). It was reported by one team that in a slightly different T2D model called the VDF rat (Vancouver Diabetic Fatty), derived from Zucker and fa/fa strains, which was used by that interventional therapy with a DPPIV inhibitor was able to partially improve glucose tolerance, peripheral insulin sensitivity, and beta-cell function (Pospisilik et al., 2002a; Pospisilik et al., 2002b). It has to be noted that this model was not diabetic, being characterized by absence of hyperglycaemia, weak glucose intolerance and no peripheral insulin resistance. It was the aim of the present invention to develop a preclinical animal model that not only better represents the progression of type-2-diabetes in humans, but that also shows stronger responses to drug therapy.
TABLE 1Main pre-clinical rodent models of metabolic diseases.Rodent models of metabolic diseases induced by diet or chemical agents.B: Rodent models of insulin resistance and T2D induced by gene defects.Diet inducedChemically-DIO ratsDIO miceSucroseinducedAHuman(SD)(AKR/J)fed ratsSTZObesity originpolygenicpolygenicpolygenicpolygenicpolygenicT2D originpolygenicpolygenicpolygenicpolygenicpolygenicLeptin/Leptin RnormalnormalnormalnormalnormalDegree of ObesityModerateSeveremoderateNoNoT2D onsetmaturematurematurematurematureElevated TGYesModerateYesYesYesHyperglycaemiaYesNoYes?Yesβ-cell failurevariableNo/???Insulin resistanceYesModerateYesYesYesHyperphagiaNoNoNoNoNoHypercorticismNoNoNoNoNoLiver steatosisVariableVariableVariable??Zuckerdb/dbBHumanfa/fa ratsZDF ratsob/ob micemiceObesity originpolygenicMonogenic:Monogenic:MonogenicMonogenicfa genefa geneT2D originpolygenicNopolygenicpolygenicpolygenicLeptin/Leptin RnormaldisrupteddisrupteddisrupteddisruptedDegree of ObesityModerateSevereSevereSevereSevereT2D onsetmatureNomatureyoungyoungElevated TGYesYesYesYesYesHyperglycaemiaYesNoYesYesYesβ-cell failurevariableNovariableNoYesInsulin resistanceYesYesYesNoYesHyperphagiaNoYesYesYesYesHypercorticismNoYesYesYesYesLiver steatosisVariable??YesYes